Isolated human tumor supressor proteins, nucleic acid molecules encoding these human tumor supressor proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of polypeptides that are encoded by genes within the human genome, the tumor supressor protein polypeptides of the present invention. The present invention specifically provides isolated polypeptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the tumor supressor protein polypeptides, and methods of identifying modulators of the tumor supressor protein polypeptides.

FIELD OF THE INVENTION

The present invention is in the field of tumor supressor proteins thatare related to the ING1 subfamily, recombinant DNA molecules and proteinproduction. The present invention specifically provides novel tumorsupressor protein polypeptides and proteins and nucleic acid moleculesencoding such peptide and protein molecules, all of which are useful inthe development of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION

Tumor supressor proteins, particularly members of the ING1 subfamilies,are a major target for drug action and development. Accordingly, it isvaluable to the field of pharmaceutical development to identify andcharacterize previously unknown members of these subfamily of tumorsupressor proteins. The present invention advances the state of the artby providing a previously unidentified human tumor supressor proteinsthat have homology to members of the ING1 subfamilies.

ING1

The candidate tumour suppressor p33ING1 cooperates with p53 in cellgrowth control. The candidate tumour-suppressor gene ING1 has beenidentified by using the genetic suppressor element (GSE) methodology.ING1 encodes a nuclear protein, p33ING1, overexpression of whichinhibits growth of different cell lines. The properties of p33ING1suggest its involvement in the negative regulation of cell proliferationand in the control of cellular ageing, anchorage dependence andapoptosis. These cellular functions depend largely on the activity ofp53, a tumour-suppressor gene that determines the cellular response tovarious types of stress. Experimental evidence indicates that thebiological effects of ING1 and p53 are interrelated and require theactivity of both genes: neither of the two genes can, on its own, causegrowth inhibition when the other is suppressed. Furthermore, activationof transcription from the p21/WAF1 promoter, a key mechanism ofp53-mediated growth control, depends on the expression of ING1. Aphysical association between p33ING1 and p53 proteins has been detectedby immunoprecipitation. These results indicate that p33ING1 is acomponent of the p53 signalling pathway that cooperates with p53 in thenegative regulation of cell proliferation by modulating p53-dependenttranscriptional activation.

Indirect immunofluorescence evidence indicatesa that the p33ING1 proteinis located in the nucleus, which is consistent with its proposed role asa growth regulator. Fluorescence in situ hybridization and radiationhybrid mapping evidence indicates that the ING1 gene is located onchromosome 13 at 13q33-q34.

Known transcripts of the ING1 gene comprise 3 exons, with 4 mRNAvariants transcribed from 3 different promoter regions. Of 34informative cases of head and neck squamous cell carcinoma, 68% oftumors showed loss of heterozygosity (LOH) at 13q33-q34, where the ING1gene is located. Gunduz et al. (Cancer Res. 60: 3143-3146 (2000) PubMedID: 10866301) found 3 missense mutations and 3 silent changes in theING1 gene in 6 of 23 tumors with allelic loss at the 13q33-q34 region.These missense mutations were found within the PHD finger domain andnuclear localization motif in the ING1 protein, probably abrogating itsnormal function. In tumor tissue of a squamous cell carcinoma of thehead and neck, Gunduz et al. (2000) found a G-to-C change (TGC-TCC) inexon 2 of the ING1 gene resulting in a cys215-to-ser substitution. Thecysteine substituted is 1 of 7 composing the C4HC3 motif of ING1. Thischange may affect the PHD finger and break the 3-dimensional structureof the ING1 protein, leading to loss of function. In tumor tissue from acase of squamous cell carcinoma of the head and neck, Gunduz et al.(2000) found a C-to-A change (GCC-GAC) in exon 2 of the ING1 generesulting in an ala192-to-asp substitution. This mutation may affect thenuclear localization signal and ultimately interfere in the accumulationof ING1 protein in the nucleus.

For more information, see Garkavtsev I, et al., Nature 391 (6664),295-298 (1998); Garkavtsev, et al., Cytogenet. Cell Genet. 76: 176-178,(1997) PubMed ID: 9186514; Garkavtsev, et al., Nature Genet. 14:415-420, (1996). Note: Erratum: Nature Genet. 23: 373 only, 1999. PubMedID: 8944021; Gunduz et al., Cancer Res. 60: 3143-3146 (2000) PubMed ID:10866301; Saito, et al., J. Hum. Genet. 45: 177-181, (2000) PubMed ID:10807544; and Zeremski, et al., Somat. Cell Molec. Genet. 23: 233-236,(1997) PubMed ID: 9330636.

p53 Protein

The gene for the nuclear phosphoprotein p53 is the most commonly mutatedgene yet identified in human cancers (Vogelstein, B., Nature, 348:681(1990)). Missense mutations occur in tumors of the colon, lung, breast,ovary, bladder, and several other organs (S. J. Baker, et al., Science,244:217 (1989); J. M. Nigro, et al., Nature, 342:705 (1989); T.Takahashi, et al., Science, 246:491 (1989); Romano, et al., Oncogene,4:1483 (1989), Menon, Proc. Natl Acad. Sci. USA, 87:5435 (1990); Iggo,et al., Lancet ii, 675 (1990); T. Takahashi, et al., J. Clin. Invest.86:363 (1990); Mulligan, Proc. Natl Acad. Sci. USA, 87:5863 (1990);Bartek, et al., Oncogene, 5:893 (1990); Stratton et al., Oncogene,5:1297 (1990)). One of the important challenges of current cancerresearch is the elucidation of the biochemical properties of the p53gene product and the way in which mutations of the p53 gene effect theseproperties.

Inactivation or loss of p53 is a common event associated with thedevelopment of human cancers. Functional inactivation may occur as aconsequence of genetic aberrations within the p53 gene, most commonlymissense mutations, or interaction with viral and cellular oncogenes.For reviews see: Levine, A. J. et al, Nature 351, 453-455 (1991),Vogelstein, B. and Kinzler, K. W., Cell 70, 523-526 (1992), Zambetti,G., and Levine, A. J., FASEB J. 7, 855-865 (1993), Harris, C. C.,Science 262 1980-1981 (1993). Loss of wild-type (wt) p53 functions leadsto uncontrolled cell cycling and replication, inefficient DNA repair,selective growth advantage and, consequently, tumor formation. Levine,A. J. et al, Nature 351, 453-455 (1991); Vogelstein, B. and Kinzler, K.W., Cell 70, 523-526 (1992); Zambetti G. and Levine, A. J., FASEB J. 7,855-865 (1993); Harris, C. C., Science 262, 1980-1981, (1993); Lane, D.P., Nature 358, 15-16 (1992); Livingstone, L. R. et al, Cell 70, 923-935(1992). Tumorigenesis may be even further accentuated by the gain of newfunctions associated with many mutant forms of p53. Chen, P. -L. et al,Science 250, 1576-1580 (1990); Dittmer, D. et al, Nature Genetics 44142-4145 (1993); Sun, Y. et al, Proc. Natl Acad. Sci. USA 90, 2827-2831(1993), providing a potential basis for their strong selection in humantumors.

Wild-type (wt) p53 is a sequence-specific DNA binding protein found inhumans and other mammals, which has tumor suppressor function (See,e.g., Harris (1993), Science, 262:1980-1981). The wild-type p53 proteinfunctions to regulate cell proliferation and cell death (also known asapoptosis). It also participates in the response of the cell to DNAdamaging agents (Harris (1993), cited above). In more than half of allhuman tumors p53 is inactivated by mutations and is therefore unable toarrest cell proliferation or induce apoptosis in response to DNAdamaging agents, such as radiation and chemotherapeutics commonly usedfor cancer treatment. The amino acid sequences of human p53 are known inthe art. (Zakut-Houri et al, (1985), EMBO J., 4:1251-1255; GenBank CodeHsp53).

At the biochemical level, p53 is a tetareric DNA sequence-specifictranscription factor. Its DNA binding and transcriptional activities arerequired for p53 to suppress tumor growth (Pietenpol et al, (1994),Proc. Natl. Acad. Sci. USA, 91:1998-2002). p53 forms homotetramers inthe absence of DNA and maintains its tetrameric stoichiometty when boundto DNA (Kraiss et al, (1988), J. Virol., 62:4737-4744; Stenger et al,(1992), Mol. Carcinog., 5:102-106; Sturzbecher et al, (1992), Oncogene,7:1513-1523; Friedman et al, (1993), Proc. Natl. Acad. Sci. USA,90:3319-3323; Halazonetis and Kandil (1993), EMBO J., 12:5057-5064; andHainaut et al, (1994), Oncogene, 9:299-303). Consistent with theobservation that p53 binds DNA as a homotetramer, the knownphysiologically relevant DNA sites recognized by p53 contain fourpentanucleotide repeats (El-DeiryDeiry et al, (1993), Cell, 75:817-825;Wu et al, (1993), Genes Dev., 7:1126-1132; Kastan et al, (1992), Cell,71:587-597). Each pentanucleotide repeat is recognized by one subunit ofthe p53 homotetramer (Halazonetis and Kandil (1993), cited above; Cho etal, (1994), Science, 265:346-355). The ability of p53 to bind DNA in asequence-specific manner has been mapped (Halazonetis and Kandil (1993),cited above; Pavletich et al, (1993), Genes Dev., 7:2556-2564; Wang etal, (1993), Genes Dev., 7:2575-2586).

Once bound to DNA, p53 activates gene transcription from neighboringpromoters. The ability of p53 to activate gene transcription has alsobeen mapped to amino acid residues 1-50 of human p53 (Fields et al,(1990), Science, 249:1046-1049).

The C-terminus of the human p53 tumor suppressor protein has twofunctions. It induces p53 oligomerization and it regulates p53 DNAbinding by controlling the conformation of p53 tetramers. These twofunctions map to independent regions. (Wang et al, (1994), Mol. Cell.Biol., 14:5182-5191; Clore et al, (1994), Science, 265:386-391).Regulation of DNA binding maps to amino acid residues 364-393 of humanp53 or to the corresponding region encompassing residues 361-390 ofmouse p53 (Hupp et al, (1992), Cell, 71:875-886; Halazonetis et al,(1993), EMBO J., 12:1021-1028; Halazonetis and Kandil (1993), citedabove; Genbank locus Mmp53r).

Mutations of the p53 protein in most human tumors involve thesequence-specific DNA binding domain, so that the mutant proteins areunable to bind DNA (Bargonetti et al, (1992), Genes Dev., 6:1886-1898).The loss of p53 function is critical for tumor development. Introductionof wild-type p53 into tumor cells leads to arrest of cell proliferationor cell death (Finlay et al, (1989), Cell, 57:1083-1093; Eliyahu et al,(1989), Proc. Natl. Acad. Sci. USA, 86:8763-8767; Baker et al, (1990),Science, 249:912-915; Mercer et al, (1990), Proc. Natl. Acad. Sci. USA,87:6166-6170; Diller et al, (1990), Mol. Cell. Biol., 10:5772-5781;Isaacs et al, (1991), Cancer Res., 51:4716-4720; Yonish-Rouach et al,(1993), Mol. Cell. Biol., 13:1415-1423; Lowe et al, (1993), Cell,74:957-967; Fujiwara et al, (1993), Cancer Res., 53:4129-4133; Fujiwaraet al, (1994), Cancer Res., 54:2287-2291).

The strong correlation between the ability of p53 to activatetranscription in a sequence specific manner and its ability to suppresscell growth or induce apoptosis Vogelstein, B. and Kinzler, K. W., Cell70 523-526 (1992), Yonish-Rouach, E. et al, Nature 352, 345-347 (1991),Lowe, S. W. et al, Nature 362, 847-849 (1993), Clark, A. R. et al,Nature 362, 849-852 (1993), Shaw, P. et al, Proc. Natl. Acad. Sci. USA89,4495-4499 (1992), suggests that p53-induced genes may play a criticalrole in mediating the function of p53 as a tumor suppressor. A fewendogenous genes have been characterized to be induced by p53. Theseinclude the mdm-2 and its human homolog hdm-2. Wu, X. et al, Genes &Development 7 1126-1132 (1993), GADD45; Kastan, M. B. et al, Cell 71,587-597 (1992);, and WAF1/CIP1/p21 El-Deiry, W. S., et al, Cell 75,817-825 (1993) genes. hdm-2 has been suggested to act as a negativefeedback regulator of p53, and in this respect would function as anoncogene. Wu, X. et al, Genes & Development 7, 1126-1132 (1993),Zambetti, G. and Levine, A. J., FASEB J. 7, 855-865 (1993). This isconsistent with amplification of the hdm-2 gene being associated withhuman cancers Oliner, J. D. et al, Nature 358, 80-83 (1992). BothWAF1/CIP1/p21, an inhibitor of cyclin-dependent kinases Harper, J. W. etal, Cell 75 805-816 (1993), Xiong, Y. et al, Nature 366, 701-704 (1993),and gadd45 (Zhan, Q. et at, Mol. Cell. Biol. 14,2361-2371 (1994)) haveso far been shown to inhibit growth of tumor cells in culture. El-Deiry,W. S. et al, Cell 75, 817-825 (1993).

The amino acid sequence of p53 is conserved across evolution (Soussi etal, (1990), Oncogene, 5:945-952), suggesting that its function is alsoconserved (See FIGS. 4 and 5). Despite this, an invertebrateortholog/homolog of known mammalian p53 protein has not yet beenidentified.

The discovery of a new human tumor suppressor proteins and thepolynucleotides which encode them satisfies a need in the art byproviding new compositions which are useful in the diagnosis,prevention, and treatment of inflammation and disorders associated withcell proliferation and apoptosis.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human tumor supressor protein polypeptides andproteins that are related to the ING1 tumor supressor protein subfamily,as well as allelic variants and other mammalian orthologs thereof. Theseunique peptide sequences; and nucleic acid sequences that encode thesepeptides, can be used as models for the development of human therapeutictargets, aid in the identification of therapeutic proteins, and serve astargets for the development of human therapeutic agents that modulatetumor supressor protein activity in cells and tissues that express thetumor supressor protein. Experimental data as provided in FIG. 1indicates expression in testis, hypothalamus, lymph, germinal center Bcells, leukocytes, and pooled germ cell tumors.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule or transcriptsequence that encodes the tumor supressor protein of the presentinvention. (SEQ ID NO:1) In addition, structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.Experimental data as provided in FIG. 1 indicates expression in testis,hypothalamus, lymph, germinal center B cells, leukocytes, and pooledgerm cell tumors.

FIG. 2 provides the predicted amino acid sequence of the tumor supressorprotein of the present invention. (SEQ ID NO:2) In addition structureand functional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the tumorsupressor protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a tumor supressor protein or part of a tumor supressorprotein and are related to the ING1 subfamily. Utilizing thesesequences, additional genomic sequences were assembled and transcriptand/or cDNA sequences were isolated and characterized. Based on thisanalysis, the present invention provides amino acid sequences of humantumor supressor protein polypeptides that are related to the ING1subfamily, nucleic acid sequences in the form of transcript sequences,cDNA sequences and/or genomic sequences that encode these tumorsupressor protein polypeptide, nucleic acid variation (allelicinformation), tissue distribution of expression, and information aboutthe closest art known protein/peptide/domain that has structural orsequence homology to the tumor supressor protein of the presentinvention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known tumor supressor proteins of the ING1subfamily and the expression pattern observed. Experimental data asprovided in FIG. 1 indicates expression in testis, hypothalamus, lymph,germinal center B cells, leukocytes, and pooled germ cell tumors. Theart has clearly established the commercial importance of members of thisfamily of proteins and proteins that have expression patterns similar tothat of the present gene. Some of the more specific features of thepeptides of the present invention, and the uses thereof, are describedherein, particularly in the Background of the Invention and in theannotation provided in the Figures, and/or are known within the art foreach of the known ING1 family or subfamily of tumor supressor proteins.

SPECIFIC EMBODIMENTS

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thetumor supressor protein family and are related to the ING1 subfamily(protein sequences are provided in FIG. 2, transcript/cDNA sequences areprovided in FIG. 1 and genomic sequences are provided in FIG. 3). Thepeptide sequences provided in FIG. 2, as well as the obvious variantsdescribed herein, particularly allelic variants as identified herein andusing the information in FIG. 3, will be referred herein as the tumorsupressor proteins or peptides of the present invention, tumor supressorproteins or peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the tumor supressor protein polypeptide disclosed in theFIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1,transcript/cDNA or FIG. 3, genomic sequence), as well as all obviousvariants of these peptides that are within the art to make and use. Someof these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components.

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thetumor supressor protein polypeptide having less than about 30% (by dryweight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

The isolated tumor supressor protein polypeptide can be purified fromcells that naturally express it, purified from cells that have beenaltered to express it (recombinant), or synthesized using known proteinsynthesis methods. Experimental data as provided in FIG. 1 indicatesexpression in testis, hypothalamus, lymph, germinal center B cells,leukocytes, and pooled germ cell tumors. For example, a nucleic acidmolecule encoding the tumor supressor protein polypeptide is cloned intoan expression vector, the expression vector introduced into a host celland the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the tumor supressor protein polypeptide of the presentinvention are the naturally occurring mature proteins. A briefdescription of how various types of these proteins can be made/isolatedis provided below.

The tumor supressor protein polypeptides of the present invention can beattached to heterologous sequences to form chimeric or fusion proteins.Such chimeric and fusion proteins comprise a tumor supressor proteinpolypeptide operatively linked to a heterologous protein having an aminoacid sequence not substantially homologous to the tumor supressorprotein polypeptide. “Operatively linked” indicates that the tumorsupressor protein polypeptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the tumor supressor protein polypeptide.

In some uses, the fusion protein does not affect the activity of thetumor supressor protein polypeptide per se. For example, the fusionprotein can include, but is not limited to, enzymatic fusion proteins,for example beta-galactosidase fusions, yeast two-hybrid GAL fusions,poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant tumor supressor protein polypeptide. In certain hostcells (e.g., mammalian host cells), expression and/or secretion of aprotein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A tumor supressor protein polypeptide-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the tumor supressor proteinpolypeptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the peptides of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art know techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the tumor supressor protein polypeptidesof the present invention. The degree of homology/identity present willbe based primarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily, and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of the reference sequence.

The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position (as used herein amino acidor nucleic acid “identity” is equivalent to amino acid or nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, word length=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the proteins ofthe invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thetumor supressor protein polypeptides of the present invention as well asbeing encoded by the same genetic locus as the tumor supressor proteinpolypeptide provided herein. As indicated by the data presented in FIG.3, the map position was determined to be on chromosome 13 by ePCR, andconfirmed with radiation hybrid mapping.

Allelic variants of a tumor supressor protein polypeptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the tumorsupressor protein polypeptide as well as being encoded by the samegenetic locus as the tumor supressor protein polypeptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 13 by ePCR, andconfirmed with radiation hybrid mapping. As used herein, two proteins(or a region of the proteins) have significant homology when the aminoacid sequences are typically at least about 70-80%, 80-90%, and moretypically at least about 90-95% or more homologous. A significantlyhomologous amino acid sequence, according to the present invention, willbe encoded by a nucleic acid sequence that will hybridize to a tumorsupressor protein polypeptide encoding nucleic acid molecule understringent conditions as more fully described below.

Paralogs of a tumor supressor protein polypeptide can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the tumor supressor proteinpolypeptide, as being encoded by a gene from humans, and as havingsimilar activity or function. Two proteins will typically be consideredparalogs when the amino acid sequences are typically at least about40-50%, 50-60%, and more typically at least about 60-70% or morehomologous through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a tumorsupressor protein polypeptide encoding nucleic acid molecule undermoderate to stringent conditions as more fully described below.

Orthologs of a tumor supressor protein polypeptide can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the tumor supressor proteinpolypeptide as well as being encoded by a gene from another organism.Preferred orthologs will be isolated from mammals, preferably primates,for the development of human therapeutic targets and agents. Suchorthologs will be encoded by a nucleic acid sequence that will hybridizeto a tumor supressor protein polypeptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the tumor supressor proteinpolypeptides of the present invention can readily be generated usingrecombinant techniques. Such variants include, but are not limited todeletions, additions and substitutions in the amino acid sequence of thetumor supressor protein polypeptide. For example, one class ofsubstitutions is conserved amino acid substitutions. Such substitutionsare those that substitute a given amino acid in a tumor supressorprotein polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr, exchange of the acidicresidues Asp and Glu, substitution between the amide residues Asn andGln, exchange of the basic residues Lys and Arg, replacements among thearomatic residues Phe, Tyr, and the like. Guidance concerning whichamino acid changes are likely to be phenotypically silent are found inBowie et al., Science 247:1306-1310 (1990).

Variant tumor supressor protein polypeptides can be fully functional orcan lack function in one or more activities. Fully functional variantstypically contain only conservative variations or variations innon-critical residues or in non-critical regions. Functional variantscan also contain substitution of similar amino acids that result in nochange or an insignificant change in function. Alternatively, suchsubstitutions may positively or negatively affect function to somedegree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro proliferative activity.Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallography, nuclearmagnetic resonance, or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992); de Vos et al Science 255:306-312 (1992)).

The present invention further provides fragments of the tumor supressorprotein polypeptides, in addition to proteins and peptides that compriseand consist of such fragments. Particularly those comprising theresidues identified in FIG. 2. The fragments to which the inventionpertains, however, are not to be construed as encompassing fragmentsthat have been disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16 or morecontiguous amino acid residues from a tumor supressor proteinpolypeptide. Such fragments can be chosen based on the ability to retainone or more of the biological activities of the tumor supressor proteinpolypeptide, or can be chosen for the ability to perform a function,e.g., act as an immunogen. Particularly important fragments arebiologically active fragments, peptides that are, for example about 8 ormore amino acids in length. Such fragments will typically comprise adomain or motif of the tumor supressor protein polypeptide, e.g., activesite. Further, possible fragments include, but are not limited to,domain or motif containing fragments, soluble peptide fragments, andfragments containing immunogenic structures. Predicted domains andfunctional sites are readily identifiable by computer programs wellknown and readily available to those of skill in the art (e.g., PROSITE,HMMer, eMOTIF, etc.). The results of one such analysis are provided inFIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in tumor supressorprotein polypeptides are described in basic texts, detailed monographs,and the research literature, and they are well known to those of skillin the art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad Sci. 663:48-62(1992)).

Accordingly, the tumor supressor protein polypeptides of the presentinvention also encompass derivatives or analogs in which a substitutedamino acid residue is not one encoded by the genetic code, in which asubstituent group is included, in which the mature tumor supressorprotein polypeptide is fused with another compound, such as a compoundto increase the half-life of the tumor supressor protein polypeptide(for example, polyethylene glycol), or in which the additional aminoacids are fused to the mature tumor supressor protein polypeptide, suchas a leader or secretory sequence or a sequence for purification of themature tumor supressor protein polypeptide, or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in assays to determinethe biological activity of the protein, including in a panel of multipleproteins for high-throughput screening; to raise antibodies or to elicitanother immune response; as a reagent (including the labeled reagent) inassays designed to quantitatively determine levels of the protein (orits ligand or receptor) in biological fluids; and as markers for tissuesin which the corresponding protein is preferentially expressed (eitherconstitutively or at a particular stage of tissue differentiation ordevelopment or in a disease state). Where the protein binds orpotentially binds to another protein (such as, for example, in areceptor-ligand interaction), the protein can be used to identify thebinding partner so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these research utilities are capableof being developed into reagent grade or kit format forcommercialization as research products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, tumor supressor proteins isolated from humansand their human/mammalian orthologs serve as targets for identifyingagents for use in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the tumor supressor protein. Experimentaldata as provided in FIG. 1 indicates that tumor supressor proteins ofthe present invention are expressed in testis, hypothalamus, lymph,germinal center B cells, leukocytes, and pooled germ cell tumors.Specifically, a virtual northern blot shows expression in testis,hypothalamus, lymph, germinal center B cells, and pooled germ celltumors. In addition, PCR-based tissue screening panel indicatesexpression in leukocytes. A large percentage of pharmaceutical agentsare being developed that modulate the activity of tumor supressorproteins, particularly members of the ING1 subfamily (see Background ofthe Invention). The structural and functional information provided inthe Background and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in testis, hypothalamus, lymph, germinalcenter B cells, leukocytes, and pooled germ cell tumors. Such uses canreadily be determined using the information provided herein, that whichis known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to tumor supressor proteins that arerelated to members of the ING1 subfamily. Such assays involve any of theknown tumor supressor protein functions or activities or propertiesuseful for diagnosis and treatment of tumor supressor protein-relatedconditions that are specific for the subfamily of tumor supressorproteins that the one of the present invention belongs to, particularlyin cells and tissues that express the tumor supressor protein.Experimental data as provided in FIG. 1 indicates that tumor supressorproteins of the present invention are expressed in testis, hypothalamus,lymph, germinal center B cells, leukocytes, and pooled germ cell tumors.Specifically, a virtual northern blot shows expression in testis,hypothalamus, lymph, germinal center B cells, and pooled germ celltumors. In addition, PCR-based tissue screening panel indicatesexpression in leukocytes.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the tumor supressor protein,as a biopsy or expanded in cell culture. Experimental data as providedin FIG. 1 indicates expression in testis, hypothalamus, lymph, germinalcenter B cells, leukocytes, and pooled germ cell tumors. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe tumor supressor protein.

The polypeptides can be used to identify compounds that modulate tumorsupressor protein activity. Both the tumor supressor protein of thepresent invention and appropriate variants and fragments can be used inhigh-throughput screens to assay candidate compounds for the ability tobind to the tumor supressor protein. These compounds can be furtherscreened against a functional tumor supressor protein to determine theeffect of the compound on the tumor supressor protein activity. Further,these compounds can be tested in animal or invertebrate systems todetermine activity/effectiveness. Compounds can be identified thatactivate (agonist) or inactivate (antagonist) the tumor supressorprotein to a desired degree.

Therefore, in one embodiment, ING1 or a fragment or derivative thereofmay be administered to a subject to prevent or treat a disorderassociated with an increase in apoptosis. Such disorders include, butare not limited to, AIDS and other infectious or geneticimmunodeficiencies, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, and cerebellar degeneration, myelodysplastic syndromes suchas aplastic anemia, ischemic injuries such as myocardial infarction,stroke, and reperfusion injury, toxin-induced diseases such asalcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseasessuch as cachexia, viral infections such as those caused by hepatitis Band C, and osteoporosis.

In another embodiment, a pharmaceutical composition comprising ING1 maybe administered to a subject to prevent or treat a disorder associatedwith increased apoptosis including, but not limited to, those listedabove.

In still another embodiment, an agonist which is specific for ING1 maybe administered to prevent or treat a disorder associated with increasedapoptosis including, but not limited to, those listed above.

In a further embodiment, a vector capable of expressing ING1, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with increased apoptosis including, but not limitedto, those listed above.

In cancer, where ING1 promotes cell proliferation, it is desirable todecrease its activity. Therefore, in one embodiment, an antagonist ofING1 may be administered to a subject to prevent or treat cancerincluding, but not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teratocarcinoma, and, in particular,cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Inone aspect, an antibody specific for ING1 may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express ING1.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding ING1 may be administered to a subject to preventor treat a cancer including, but not limited to, the types of cancerlisted above.

In inflammation, where ING1 promotes cell proliferation, it is desirableto decrease its activity. Therefore, in one embodiment, an antagonist ofING1 may be administered to a subject to prevent or treat aninflammation. Disorders associated with inflammation include, but arenot limited to, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, atrophic gastritis, glomenilonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma. In one aspect, anantibody specific for ING1 may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express ING1.

Further, the tumor supressor protein polypeptides can b used to screen acompound for the ability to stimulate or inhibit interaction between thetumor supressor protein and a molecule that normally interacts with thetumor supressor protein, e.g. a ligand or a component of the signalpathway that the tumor supressor protein normally interacts. Such assaystypically include the steps of combining the tumor supressor proteinwith a candidate compound under conditions that allow the tumorsupressor protein, or fragment, to interact with the target molecule,and to detect the formation of a complex between the protein and thetarget or to detect the biochemical consequence of the interaction withthe tumor supressor protein and the target, such as any of theassociated effects of signal transduction.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries). (Hodgson, Bio/technology, Sep. 10,1992(9);973-80).

One candidate compound is a soluble fragment of the tumor supressorprotein that competes for ligand binding. Other candidate compoundsinclude mutant tumor supressor proteins or appropriate fragmentscontaining mutations that affect tumor supressor protein function andthus compete for ligand. Accordingly, a fragment that competes forligand, for example with a higher affinity, or a fragment that bindsligand but does not allow release, is within the scope of the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) tumor supressor proteinactivity. The assays typically involve an assay of events in the tumorsupressor protein mediated signal transduction pathway that indicatetumor supressor protein activity. Thus, the phosphorylation of aprotein/ligand target, the expression of genes that are up- ordown-regulated in response to the tumor supressor protein dependentsignal cascade can be assayed. In one embodiment, the regulatory regionof such genes can be operably linked to a marker that is easilydetectable, such as luciferase. Alternatively, phosphorylation of thetumor supressor protein, or a tumor supressor protein target, could alsobe measured.

Any of the biological or biochemical functions mediated by the tumorsupressor protein can be used as an endpoint assay. These include all ofthe biochemical or biochemical/biological events described herein, inthe references cited herein, incorporated by reference for theseendpoint assay targets, and other functions known to those of ordinaryskill in the art.

Binding and/or activating compounds can also be screened by usingchimeric tumor supressor proteins in which any of the protein's domains,or parts thereof, can be replaced by heterologous domains or subregions.Accordingly, a different set of signal transduction components isavailable as an end-point assay for activation. This allows for assaysto be performed in other than the specific host cell from which thetumor supressor protein is derived.

The tumor supressor protein polypeptide of the present invention is alsouseful in competition binding assays in methods designed to discovercompounds that interact with the tumor supressor protein. Thus, acompound is exposed to a tumor supressor protein polypeptide underconditions that allow the compound to bind or to otherwise interact withthe polypeptide. Soluble tumor supressor protein polypeptide is alsoadded to the mixture. If the test compound interacts with the solubletumor supressor protein polypeptide, it decreases the amount of complexformed or activity from the tumor supressor protein target. This type ofassay is particularly useful in cases in which compounds are sought thatinteract with specific regions of the tumor supressor protein. Thus, thesoluble polypeptide that competes with the target tumor supressorprotein region is designed to contain peptide sequences corresponding tothe region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the tumor supressor protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase/15625 fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level oftumor supressor protein-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin withtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a tumor supressor protein-binding protein and a candidate compoundare incubated in the tumor supressor protein-presenting wells and theamount of complex trapped in the well can be quantitated. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the tumor supressor protein target molecule, orwhich are reactive with tumor supressor protein and compete with thetarget molecule, as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

Agents that modulate one of the tumor supressor proteins of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal/insectmodel system. Such model systems are well known in the art and canreadily be employed in this context.

Modulators of tumor supressor protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the tumor supressor protein associated pathway, bytreating cells that express the tumor supressor protein. Experimentaldata as provided in FIG. 1 indicates expression in testis, hypothalamus,lymph, germinal center B cells, leukocytes, and pooled germ cell tumors.These methods of treatment include the steps of administering themodulators of protein activity in a pharmaceutical composition asdescribed herein, to a subject in need of such treatment.

In yet another aspect of the invention, the tumor supressor proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232(1993);. Madura et al., J. Biol. Chem. 268:12046-12054 (1993); Bartel etal., Biotechniques 14:920-924 (1993); Iwabuchi et al., Oncogene8:1693-1696 (1993); and Brent WO94/10300), to identify other proteinsthat bind to or interact with the tumor supressor protein and areinvolved in tumor supressor protein activity. Such tumor supressorprotein-binding proteins are also likely to be involved in thepropagation of signals by the tumor supressor proteins or tumorsupressor protein targets as, for example, downstream elements of atumor supressor protein-mediated signaling pathway, e.g., a painsignaling pathway. Alternatively, such tumor supressor protein-bindingproteins are likely to be tumor supressor protein inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a tumor supressorprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a tumorsupressor protein-dependent complex, the DNA-binding and activationdomains of the transcription factor are brought into close proximity.This proximity allows transcription of a reporter gene (e.g., LacZ)which is operably linked to a transcriptional regulatory site responsiveto the transcription factor. Expression of the reporter gene can bedetected and cell colonies containing the functional transcriptionfactor can be isolated and used to obtain the cloned gene which encodesthe protein which interacts with the tumor supressor protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a tumor supressor protein modulating agent, anantisense tumor supressor protein nucleic acid molecule, a tumorsupressor protein-specific antibody, or a tumor supressorprotein-binding partner) can be used in an animal or insect model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or insect model to determine the mechanism of actionof such an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

The tumor supressor proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to a diseasemediated by the peptide, Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in testis, hypothalamus, lymph, germinal center Bcells, leukocytes, and pooled germ cell tumors. The method involvescontacting a biological sample with a compound capable of interactingwith the receptor protein such that the interaction can be detected.Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells, and fluids present within a subject.

The peptides also are useful to provide a target for diagnosing adisease or predisposition to a disease mediated by the peptide,Accordingly, the invention provides methods for detecting the presence,or levels of, the protein in a cell, tissue, or organism. The methodinvolves contacting a biological sample with a compound capable ofinteracting with the receptor protein such that the interaction can bedetected.

The peptides of the present invention also provide targets fordiagnosing active disease, or predisposition to a disease, in a patienthaving a variant peptide. Thus, the peptide can be isolated from abiological sample and assayed for the presence of a genetic mutationthat results in translation of an aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered receptor activity in cell-basedor cell-free assay, alteration in ligand or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence using a detection reagents, such as an antibody orprotein binding agent.. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques. Particularly useful are methods that detectthe allelic variant of a peptide expressed in a subject and methodswhich detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the receptor protein in which one ormore of the receptor functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other ligand-binding regions that are moreor less active in ligand binding, and receptor activation. Accordingly,ligand dosage would necessarily be modified to maximize the therapeuticeffect within a given population containing a polymorphism. As analternative to genotyping, specific polymorphic peptides could beidentified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in testis,hypothalamus, lymph, germinal center B cells, leukocytes, and pooledgerm cell tumors. Accordingly, methods for treatment include the use ofthe tumor supressor protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe tumor supressor proteins. Antibodies can be prepared from any regionof the peptide as described herein. However, preferred regions willinclude those involved in function/activity and/or receptor/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection of an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates thattumor supressor proteins of the present invention are expressed intestis, hypothalamus, lymph, germinal center B cells, leukocytes, andpooled germ cell tumors. Specifically, a virtual northern blot showsexpression in testis, hypothalamus, lymph, germinal center B cells, andpooled germ cell tumors. In addition, PCR-based tissue screening panelindicates expression in leukocytes. Further, such antibodies can be usedto detect protein in situ, in vitro, or in a cell lysate or supernatantin order to evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development. Antibody detection ofcirculating fragments of the full-length protein can be used to identifyturnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in testis, hypothalamus, lymph, germinal center B cells,leukocytes, and pooled germ cell tumors. If a disorder is characterizedby a specific mutation in the protein, antibodies specific for thismutant protein can be used to assay for the presence of the specificmutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in testis,hypothalamus, lymph, germinal center B cells, leukocytes, and pooledgerm cell tumors. The diagnostic uses can be applied, not only ingenetic testing, but also in monitoring a treatment modality.Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the or relevant fragments can be used to monitor therapeuticefficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in testis, hypothalamus, lymph,germinal center B cells, leukocytes, and pooled germ cell tumors. Thus,where a specific protein has been correlated with expression in aspecific tissue, antibodies that are specific for this protein can beused to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the tumor supressor protein to abinding partner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a tumor supressor protein polypeptide of the presentinvention. Such nucleic acid molecules will consist of, consistessentially of, or comprise a nucleotide sequence that encodes one ofthe tumor supressor protein polypeptides of the present invention, anallelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB,particularly contiguous peptide encoding sequences and peptide encodingsequences within the same gene but separated by introns in the genomicsequence. The important point is that the nucleic acid is isolated fromremote and unimportant flanking sequences such that it can be subjectedto the specific manipulations described herein such as recombinantexpression, preparation of probes and primers, and other uses specificto the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. However, the nucleic acidmolecule can be fused to other coding or regulatory sequences and stillbe considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule. The present invention further provides nucleic acidmolecules that consist essentially of the nucleotide sequence shown inFIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomicsequence), or any nucleic acid molecule that encodes the proteinprovided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consistsessentially of a nucleotide sequence when such a nucleotide sequence ispresent with only a few additional nucleic acid residues in the finalnucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

Full-length genes may be cloned from known sequence using any one of anumber of methods known in the art. For example, a method which employsXL-PCR (Perkin-Elmer, Foster City, Calif.) to amplify long pieces of DNAmay be used. Other methods for obtaining full-length sequences are wellknown in the art.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life, or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the tumor supressor proteinpolypeptide alone, the sequence encoding the mature peptide andadditional coding sequences, such as a leader or secretory sequence(e.g., a pre-pro or pro-protein sequence), the sequence encoding themature peptide, with or without the additional coding sequences, plusadditional non-coding sequences, for example introns and non-coding 5′and 3′ sequences such as transcribed but non-translated sequences thatplay a role in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding, and stability of mRNA. Inaddition, the nucleic acid molecule may be fused to a marker sequenceencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form of DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention and that encodeobvious variants of the tumor supressor proteins of the presentinvention that are described above. Such nucleic acid molecules may benaturally occurring, such as allelic variants (same locus), paralogs(different locus), and orthologs (different organism), or may beconstructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to nucleic acid molecules, cells, or wholeorganisms. Accordingly, as discussed above, the variants can containnucleotide substitutions, deletions inversions, and/or insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in the FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences, and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could be at least 30, 40, 50, 100250, or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encodeepitope-bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50, or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. As indicated by the data presentedin FIG. 3, the map position was determined to be on chromosome 13 byePCR, and confirmed with radiation hybrid mapping.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asthose, which may encompass fragments disclosed prior to the presentinvention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 13 by ePCR, andconfirmed with radiation hybrid mapping.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.Moreover, the nucleic acid molecules are useful for constructingtransgenic animals wherein a homolog of the nucleic acid molecule hasbeen “knocked-out” of the animal's genome.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form, and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates that tumorsupressor proteins of the present invention are expressed in testis,hypothalamus, lymph, germinal center B cells, leukocytes, and pooledgerm cell tumors. Specifically, a virtual northern blot shows expressionin testis, hypothalamus, lymph, germinal center B cells, and pooled germcell tumors. In addition, PCR-based tissue screening panel indicatesexpression in leukocytes. Accordingly, the probes can be used to detectthe presence of, or to determine levels of, a specific nucleic acidmolecule in cells, tissues, and in organisms. The nucleic acid whoselevel is determined can be DNA or RNA. Accordingly, probes correspondingto the peptides described herein can be used to assess expression and/orgene copy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease intumor supressor protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a tumor supressor protein, such as bymeasuring a level of a receptor-encoding nucleic acid in a sample ofcells from a subject e.g., mRNA or genomic DNA, or determining if areceptor gene has been mutated. Experimental data as provided in FIG. 1indicates that tumor supressor proteins of the present invention areexpressed in testis, hypothalamus, lymph, germinal center B cells,leukocytes, and pooled germ cell tumors. Specifically, a virtualnorthern blot shows expression in testis, hypothalamus, lymph, germinalcenter B cells, and pooled germ cell tumors. In addition, PCR-basedtissue screening panel indicates expression in leukocytes.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate tumor supressor protein nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe tumor supressor protein gene, particularly biological andpathological processes that are mediated by the tumor supressor proteinin cells and tissues that express it. Experimental data as provided inFIG. 1 indicates expression in testis, hypothalamus, lymph, germinalcenter B cells, leukocytes, and pooled germ cell tumors. The methodtypically includes assaying the ability of the compound to modulate theexpression of the tumor supressor protein nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired tumor supressor protein nucleic acidexpression. The assays can be performed in cell-based and cell-freesystems. Cell-based assays include cells naturally expressing the tumorsupressor protein nucleic acid or recombinant cells geneticallyengineered to express specific nucleic acid sequences.

The assay for tumor supressor protein nucleic acid expression caninvolve direct assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thetumor supressor protein signal pathway can also be assayed. In thisembodiment the regulatory regions of these genes can be operably linkedto a reporter gene such as luciferase.

Thus, modulators of tumor supressor protein gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof tumor supressor protein mRNA in the presence of the candidatecompound is compared to the level of expression of tumor supressorprotein mRNA in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate tumor supressor protein nucleic acidexpression in cells and tissues that express the tumor supressorprotein. Experimental data as provided in FIG. 1 indicates that tumorsupressor proteins of the present invention are expressed in testis,hypothalamus, lymph, germinal center B cells, leukocytes, and pooledgerm cell tumors. Specifically, a virtual northern blot shows expressionin testis, hypothalamus, lymph, germinal center B cells, and pooled germcell tumors. In addition, PCR-based tissue screening panel indicatesexpression in leukocytes. Modulation includes both up-regulation (i.e.activation or agonization) or down-regulation (suppression orantagonization) of nucleic acid expression.

Alternatively, a modulator for tumor supressor protein nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the tumor supressor protein nucleic acid expression in thecells and tissues that express the protein. Experimental data asprovided in FIG. 1 indicates expression in testis, hypothalamus, lymph,germinal center B cells, leukocytes, and pooled germ cell tumors.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe tumor supressor protein gene in clinical trials or in a treatmentregimen. Thus, the gene expression pattern can serve as a barometer forthe continuing effectiveness of treatment with the compound,particularly with compounds to which a patient can develop resistance.The gene expression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in tumor supressor protein nucleic acid, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in tumor supressorprotein genes and gene expression products such as mRNA. The nucleicacid molecules can be used as hybridization probes to detect naturallyoccurring genetic mutations in the tumor supressor protein gene andthereby to determine whether a subject with the mutation is at risk fora disorder caused by the mutation. Mutations include deletion, addition,or substitution of one or more nucleotides in the gene, chromosomalrearrangement, such as inversion or transposition, modification ofgenomic DNA, such as aberrant methylation patterns, or changes in genecopy number, such as amplification. Detection of a mutated form of thetumor supressor protein gene associated with a dysfunction provides adiagnostic tool for an active disease or susceptibility to disease whenthe disease results from overexpression, underexpression, or alteredexpression of a tumor supressor protein.

Individuals carrying mutations in the tumor supressor protein gene canbe detected at the nucleic acid level by a variety of techniques. Asindicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 13 by ePCR, and confirmed with radiationhybrid mapping. Genomic DNA can be analyzed directly or can be amplifiedby using PCR prior to analysis. RNA or cDNA can be used in the same way.In some uses, detection of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al., NucleicAcids Res. 23:675-682 (1995)). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

Alternatively, mutations in a tumor supressor protein gene can bedirectly identified, for example, by alterations in restriction enzymedigestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutanttumor supressor protein gene and a wild-type gene can be determined bydirect DNA sequencing. A variety of automated sequencing procedures canbe utilized when performing the diagnostic assays (Naeve, C. W.,Biotechniques 19:448 (1995)), including sequencing by mass spectrometry(see, e.g., PCT International Publication No. WO 94/16101; Cohen et al.,Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal, Mutat. Res. 285:125-144 (1993); and Hayashi et a., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include, selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the tumor supressorprotein gene in an individual in order to select an appropriate compoundor dosage regimen for treatment.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol tumor supressor protein gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of tumor supressorprotein. An antisense RNA or DNA nucleic acid molecule would hybridizeto the mRNA and thus block translation of mRNA into tumor supressorprotein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of tumor supressor protein nucleicacid. Accordingly, these molecules can treat a disorder characterized byabnormal or undesired tumor supressor protein nucleic acid expression.This technique involves cleavage by means of ribozymes containingnucleotide sequences complementary to one or more regions in the mRNAthat attenuate the ability of the mRNA to be translated. Possibleregions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of thetumor supressor protein, such as ligand binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in tumor supressor proteingene expression. Thus, recombinant cells, which include the patient'scells that have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired tumorsupressor protein to treat the individual.

The invention also encompasses kits for detecting the presence of atumor supressor protein nucleic acid in a biological sample.Experimental data as provided in FIG. 1 indicates that tumor supressorproteins of the present invention are expressed in testis, hypothalamus,lymph, germinal center B cells, leukocytes, and pooled germ cell tumors.Specifically, a virtual northern blot shows expression in testis,hypothalamus, lymph, germinal center B cells, and pooled germ celltumors. In addition, PCR-based tissue screening panel indicatesexpression in leukocytes. For example, the kit can comprise reagentssuch as a labeled or labelable nucleic acid or agent capable ofdetecting tumor supressor protein nucleic acid in a biological sample;means for determining the amount of tumor supressor protein nucleic acidin the sample; and means for comparing the amount of tumor supressorprotein nucleic acid in the sample with a standard. The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect tumor supressorprotein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides arrays or microarrays of nucleicacid molecules that are based on the sequence information provided inFIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application W095/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet. al., U.S. Pat. No. 5,807,522.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides that are only 7-20 nucleotidesin length. The microarray may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull-length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene(s) of interest (or an ORF identified from thecontigs of the present invention) is typically examined using a computeralgorithm that starts at the 5′ or at the 3′ end of the nucleotidesequence. Typical algorithms will then identify oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide that preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application W095/251 116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray, the RNA or DNAfrom a biological sample is made into hybridization probes. The mRNA isisolated, and cDNA is produced and used as a template to make antisenseRNA (aRNA). The aRNA is amplified in the presence of fluorescentnucleotides, and labeled probes are incubated with the microarray sothat the probe sequences hybridize to complementary oligonucleotides ofthe microarray. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray. The biological samples may be obtained from any bodilyfluids (such as blood, urine, saliva, phlegm, gastric juices, etc.),cultured cells, biopsies, or other tissue preparations. A detectionsystem may be used to measure the absence, presence, and amount ofhybridization for all of the distinct sequences simultaneously. Thisdata may be used for large-scale correlation studies on the sequences,expression patterns, mutations, variants, or polymorphisms amongsamples.

Using such arrays, the present invention provides methods to identifythe expression of one or more of the proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid. Preferred kits will include chips that are capable of detectingthe expression of 10 or more, 100 or more, or 500 or more, 1000 or more,or all of the genes expressed in Human.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified tumor supressor protein genes of the presentinvention can be routinely identified using the sequence informationdisclosed herein can be readily incorporated into one of the establishedkit formats which are well known in the art, particularly expressionarrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in procaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning. A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterotumor supressor protein. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in a host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943 (1982)), pJRY88 (Schultzet al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance, propagation, or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced, or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as kinases,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with kinases, the protein can be isolated from the host cell bystandard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a tumorsupressor protein polypeptide that can be further purified to producedesired amounts of tumor supressor protein or fragments. Thus, hostcells containing expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe tumor supressor protein or tumor supressor protein fragments. Thus,a recombinant host cell expressing a native tumor supressor protein isuseful for assaying compounds that stimulate or inhibit tumor supressorprotein function.

Host cells are also useful for identifying tumor supressor proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutanttumor supressor protein (for example, stimulating or inhibitingfunction) which may not be indicated by their effect on the native tumorsupressor protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a tumor supressorprotein and identifying and evaluating modulators of tumor supressorprotein activity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the tumor supressor proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the tumor supressor protein toparticular cells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(O) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,tumor supressor protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo tumorsupressor protein function, including ligand interaction, the effect ofspecific mutant tumor supressor proteins on tumor supressor proteinfunction and ligand interaction, and the effect of chimeric tumorsupressor proteins. It is also possible to assess the effect of nullmutations, which is mutations that substantially or completely eliminateone or more tumor supressor protein functions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention, which are obvious to those skilled inthe field of molecular biology or related fields, are intended to bewithin the scope of the following claims.

1. An isolated polypeptide consisting of an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence shown in SEQ IDNO:2; (b) an amino acid sequence of an allelic variant of an amino acidsequence shown in SEQ ID NO:2, wherein said allelic variant is encodedby a nucleic acid molecule that hybridizes under stringent conditions tothe opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or3; (c) an amino acid sequence of an ortholog of an amino acid sequenceshown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acidmolecule that hybridizes under stringent conditions to the oppositestrand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) afragment of an amino acid sequence shown in SEQ ID NO:2, wherein saidfragment comprises at least 10 contiguous amino acids.
 2. An isolatedpolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) anamino acid sequence of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said allelic variant is encoded by anucleic acid molecule that hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(c) an amino acid sequence of an ortholog of an amino acid sequenceshown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acidmolecule that hybridizes under stringent conditions to the oppositestrand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) afragment of an amino acid sequence shown in SEQ ID NO:2, wherein saidfragment comprises at least 10 contiguous amino acids.
 3. An isolatedantibody that selectively binds to a polypeptide of claim
 2. 4. Anisolated nucleic acid molecule consisting of a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 5. An isolated nucleicacid molecule comprising a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence that encodes an amino acidsequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes ofan allelic variant of an amino acid sequence shown in SEQ ID NO:2,wherein said nucleotide sequence hybridizes under stringent conditionsto the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1or 3; (c) a nucleotide sequence that encodes an ortholog of an aminoacid sequence shown in SEQ ID NO:2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO:2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the polypeptides of claim1 comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the polypeptides are expressed from thenucleotide sequence.
 11. A method for producing any of the polypeptidesof claim 2 comprising introducing a nucleotide sequence encoding any ofthe amino acid sequences in (a)-(d) into a host cell, and culturing thehost cell under conditions in which the polypeptides are expressed fromthe nucleotide sequence.
 12. A method for detecting the presence of anyof the polypeptides of claim 2 in a sample, said method comprisingcontacting said sample with a detection agent that specifically allowsdetection of the presence of the polypeptide in the sample and thendetecting the presence of the polypeptide.
 13. A method for detectingthe presence of a nucleic acid molecule of claim 5 in a sample, saidmethod comprising contacting the sample with an oligonucleotide thathybridizes to said nucleic acid molecule under stringent conditions anddetermining whether the oligonucleotide binds to said nucleic acidmolecule in the sample.
 14. A method for identifying a modulator of apolypeptide of claim 2, said method comprising contacting saidpolypeptide with an agent and determining if said agent has modulatedthe function or activity of said polypeptide.
 15. The method of claim14, wherein said agent is administered to a host cell comprising anexpression vector that expresses said polypeptide.
 16. A method foridentifying an agent that binds to any of the polypeptides of claim 2,said method comprising contacting the polypeptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the polypeptide.
 17. A pharmaceuticalcomposition comprising an agent identified by the method of claim 16 anda pharmaceutically acceptable carrier therefor.
 18. A method fortreating a disease or condition mediated by a human tumor supressorprotein, said method comprising administering to a patient apharmaceutically effective amount of an agent identified by the methodof claim
 16. 19. A method for identifying a modulator of the expressionof a polypeptide of claim 2, said method comprising contacting a cellexpressing said polypeptide with an agent, and determining if said agenthas modulated the expression of said polypeptide.
 20. An isolated humantumor supressor protein polypeptide having an amino acid sequence thatshares at least 70% homology with an amino acid sequence shown in SEQ IDNO:2.
 21. A polypeptide according to claim 20 that shares at least 90percent homology with an amino acid sequence shown in SEQ ID NO:2. 22.An isolated nucleic acid molecule encoding a human tumor supressorprotein polypeptide, said nucleic acid molecule sharing at least 80percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or3.
 23. A nucleic acid molecule according to claim 22 that shares atleast 90 percent homology with a nucleic acid molecule shown in SEQ IDNOS:1 or 3.